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Abstract

Pollen tube is regarded as an excellent single-cell model system in plant cell studies. This protocol describes the use of a rapid and reliable immunofluorescence labeling method for studying in situ localization of proteins in pollen tubes. The whole experiment contains two major steps: pollen tube in vitro germination, and pollen tube fixation and immunolabeling. It takes about 2 days from pollen tube germination to immunofluorescence detection.

Materials and Reagents

Lily, tobacco or Arabidopsis pollen tubes Note: Generally, mature lily and tobacco pollen grains are harvested 1-2 days after they are completely released from the anthers of flowers (Wang et al., 2010). For Arabidopsis, day-0 flowers are used for pollen collection (Boavida and McCormick, 2007).

Spin down the fixed pollen tubes at 1,000 rpm for 3 min at RT using free angle rotor. Sample washing procedure: Remove the supernatant and wash the pollen tubes by adding 2 ml pollen germination medium. Gently resuspend pollen tubes by mixing them in the medium by end-to-end rotating the tube by hands. Then keep the pollen tubes on bench undisturbed and wait for them naturally sink to the bottom of the tube at RT (repeat four times).Note: All the washing steps afterwards are performed as the same with step B2.

Wash the pollen tubes with B2 for three times and keep the samples in B2 in the dark without shaking (first time: 15 min, second time: 15 min, third time: 20 min).

Add about 150 μl sample cells in confocal dish for confocal observation. Figure 1 shows the representative confocal images showing the results of immunofluorscences labeling of tobacco pollen tubes with anti-VSR antibodies (Figure 1A) and control labeling using anti-GFP antibodies (Figure 1B). The confocal fluorescence images were collected using a Leica TCS SP8 system with the following parameters: 63x water objective, 2x zoom, 900 gain, 0 background, 0.168 µm pixel size, photomultiplier tubes (PMTs) detector. The images from pollen tubes labeled with antibodies were collected with a laser level of ≤3% to ensure that the fluorescence signal was within the linear range of detection (typically 0.5 or 1% laser was used).

The original version of this protocol was described in Wang et al. (2010). This work was supported by grants from the Research Grants Council of Hong Kong (CUHK 466011, 465112, 466613 and CUHK2/CRF/11G) to L.J.

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